Investigation of the polyamine biosynthetic and transport capability of Streptococcus agalactiae: the non-essential PotABCD transporter

Polyamines constitute a group of organic polycations positively charged at physiological pH. They are involved in a large variety of biological processes, including the protection against physiological stress. In this study, we show that the genome of Streptococcus agalactiae , a commensal bacterium of the intestine and the vagina and one of the most common agents responsible of neonate infections, does not encode proteins homologous to the specific enzymes involved in the known polyamine synthetic pathways. This lack of biosynthetic capability was verified experimentally by TLC analysis of the intracellular content of S. agalactiae grown in the absence of polyamines. However, similar analyses showed that the polyamines spermidine, spermine and putrescine can be imported from the growth media into the bacteria. We found that all strains of S. agalactiae possess the genes encoding the polyamine ABC transporter PotABCD. We demonstrated that these genes form an operon with folK, a gene involved in folate biosynthesis, murB, a gene involved in peptidoglycan biosynthesis, and with clc, a gene encoding a Cl−/H+ antiporter involved in resistance to acid stress in Escherichia coli . Transcription of the potABCD operon is induced by peroxide-induced oxidative stress but not by acidic stress. Spermidine and spermine were found to be inducers of potABCD transcription at pH 7.4 whereas putrescine induces this expression only during peroxide-induced oxidative stress. Using a deletion mutant of potABCD, we were nevertheless unable to associate phenotypic traits to the PotABCD transporter, probably due to the existence of one or more as yet identified transporters with a redundant action.


INTRODUCTION
Polyamines, small aliphatic hydrocarbon molecules with a quaternary nitrogen chemical group, have a net positive charge at physiological pH. They are associated with a large range of biological functions such as efficient DNA replication, transcription, translation, stress resistance, cell proliferation and differentiation [1,2]. Together with Mg 2+ and Ca 2+ , polyamines constitute the major polycations in cells. They are able to bind to intracellular polyanions such as nucleic acids and ATP to modulate their functions. Putrescine (1,4-diaminobutane), spermidine [N-(3-aminopropyl)butane-1,4-diamine], spermine [N,N′-bis (3-aminopropyl)butane-1,4-diamine] and cadaverine (1,5-diaminopentane) are the most widely distributed cellular polyamines and are essential for normal multiplication and cellular growth of most prokaryotic and eukaryotic cells [2]. Polyamines appear to play a crucial role in the pathogenesis and virulence of important human bacterial pathogens. Several studies, in species such as Escherichia coli and Streptococcus pneumoniae, involved polyamines in the protection of bacterial cells from the toxic effects of reactive oxygen, by their function of radical scavengers [3,4]. In addition, polyamines are key mediators in the resistance to acidic stress in several bacterial species. For example, in E. coli and Salmonella enterica, they induce the expression of amino acid decarboxylases, which are directly involved in the response of these bacteria to acidic stress, and thus facilitate their survival in vivo [5,6]. The pleiotropic effects of polyamines on nucleic acid stability, transcription and translation also play an important role in the physiological adaptation of S. pneumoniae during temperature stress [4]. The physiological functions of polyamines cannot take place without a highly regulated level of intracellular polyamines, which is based on OPEN ACCESS the coordination of the processes of the polyamine uptake, synthesis and degradation [2]. Most prokaryotes have de novo biosynthesis pathways in which polyamines are generated via enzymatic modification of amino acid precursors [7]. In addition, almost all bacteria possess polyamine transport systems to satisfy their requirements from the environment [8].
Three pathways of putrescine biosynthesis (Ip, IIp and IIIp; Fig. 1) and three pathways of spermidine biosynthesis (Is, IIs and IIIs; Fig. 1) have been described in bacteria [9][10][11][12][13][14][15][16]. In Saccharomyces cerevisiae, spermine can be synthesized by the addition of a propylamine group to spermidine, a reaction catalysed by spermine synthase (Fig. 1) [17]. However, although the presence of spermine is attested to in several bacterial species, no specific bacterial spermine synthase has yet been discovered [18]. Polyamines are not biosynthesized by all bacterial species. For example, in the genus Streptococcus, while polyamine biosynthesis pathways (IIp, Is and IIs) are present in all strains of S. pneumoniae, they are lacking in the majority of the strains of Streptococus suis and Streptococcus mitis, which must acquire polyamines from the environment [19].
Almost all bacteria can also import extracellular polyamines by a polyamine ATP-binding cassette (ABC) transporter, encoded by an operon of four genes [4,20,21]. Some bacteria, such as E. coli, possess two polyamine ABC transporters, PotABCD and PotFGHI, having a higher affinity for either spermidine or putrescine, respectively [22,23]. Only a single potABCD operon is detected in many other bacteria, such as Staphylococcus aureus, S. pneumoniae, S. suis or Streptococcus agalactiae [19,[24][25][26][27]. PotA and PotG are membrane-associated cytosolic ATPases. PotB with PotC or PotH with PotI constitute transmembrane channels for polyamine transport. Located in the periplasm or anchored to the cytoplasmic membrane, PotD and PotF are substrate-binding proteins that trap extracellular polyamines [22,23]. The binding of polyamine to the substratebinding proteins results in a conformational change of the membrane spanning proteins of the transporter, which leads to ATP hydrolysis and polyamine uptake. The presence of excess polyamines in the environment can function as a feedback regulator on polyamine transport. It was shown in E. coli that a high concentration of spermidine can inhibit the polyamine transport system, by the inhibition of ATPase activity, through the interaction of spermidine with a domain of PotA [28]. In addition, PotD is also able to inhibit the transcription of the potABCD operon of E. coli [8].
S. agalactiae, also called Group B Streptococcus, was first distinguished from other streptococci by Rebecca Lancefield in 1930, after it was isolated from milk, and was detected as a primordial cause of mastitis in cows [29]. It is a Gram-positive, β-haemolytic bacterium, which frequently and asymptomatically colonizes the gastrointestinal and/ or urogenital tract of humans [30]. S. agalactiae maternal carriage was identified as a high risk factor for the development of neonatal disease and preterm birth [31]. In neonates, S. agalactiae is one of the leading causes of invasive infections, such as pneumonia, septicaemia and meningitis [32]. It has also emerged as an increasingly common cause of invasive diseases in immunocompromised and elderly adults [33]. In addition to its ability to colonize the gastrointestinal and uro-genital tracts, S. agalactiae also colonizes the throat or the oral and nasopharyngeal mucosa. Furthermore, it is able to infect the amniotic and cerebrospinal fluids, the blood and the mammary gland [34][35][36][37]. Moreover, S. agalactiae can contaminate foodstuffs, and it has been isolated from pastries and seafood products [38]. Such ability to survive in many different environments indicates its large capability for adaptation. As polyamines are involved in the resistance mechanism of several bacterial species to environmental stress, they could be involved in the substantial ability for adaptation of S. agalactiae. However, the physiological role of polyamines and the phenotypic traits given by the PotABCD transporter have not yet been studied in S. agalactiae.
In the genome of all Streptococcus species, a gene called murB, which encodes an enzyme implicated in peptidoglycan biosynthesis, is localized upstream of the potABCD operon [19]. We previously identified the transcriptional promoter of murB in the folK-murB intergenic region of S. agalactiae. We also localized another transcriptional promoter in the upstream folK gene and we showed that genes involved in folate biosynthesis are co-transcribed with murB. This co-transcription could be necessary to synchronize two processes of cell wall synthesis, as it was postulated that a folic acid-mediated reaction might be involved in cell wall synthesis [39,40]. In some S. agalactiae strains, the mobile element IS1548 is inserted in the folK-murB intergenic region. The presence of this insertion sequence prevents the co-transcription of murB with genes of the folate pathway. However, as IS1548 brings an additional promoter able to initiate murB transcription, the insertion of IS1548 results in a minor negative modulation of the expression of murB [40]. In S. pneumoniae and S. suis, it was demonstrated that murB is co-transcribed with potABCD, which suggests also a relationship between polyamine transport and peptidoglycan biosynthesis in streptococci [19,41]. Polyamines were described to be components of the peptidoglycan. In S. pneumoniae, putrescine can substitute for choline, which is involved in peptidoglycan synthesis and hydrolysis [41]. In S. suis, peptidoglycan synthesis and separation of daughter cells during cell division cannot be completed without the presence of polyamines [19]. In Gram-negative bacteria, spermidine, putrescine and cadaverine are also considered as constituents of the peptidoglycan, since they play a significant role in maintaining cell shape and integrity of the cell surface structure [42,43]. In S. agalactiae, the clc gene is located downstream of the potABCD genes. The murB, potABCD and clc genes are all transcribed in the same direction. The clc gene is predicted to code a Cl − /H + antiporter [27]. Cl − /H + antiporters found in E. coli promote proton expulsion and were described to be highly induced under acid stress [44]. The presence of clc downstream of potABCD is noteworthy since, as discussed above, polyamines are involved in the resistance of some bacteria to acidic stress.
In this study, we first analysed the genome of S. agalactiae strains available at the National Center for Biotechnology Information (NCBI) database to look for the presence of genes encoding enzymes involved in known spermine, spermidine and putrescine biosynthesis pathways. Similarly, the prevalence of the potABCD operon in S. agalactiae strains was evaluated by blast analysis of completely sequenced genomes. The polyamine biosynthetic and transport capability of S. agalactiae was tested by TLC analyses of the intracellular polyamine content of bacteria grown in the absence or in the presence of polyamines. We then examined the transcription pattern of the potABCD region and analysed the expression of the pot operon in response to polyamines and various stress conditions. The influence of polyamines on the growth and on the survival of S. agalactiae was finally determined.

Plasmids, bacterial strains and growth conditions
The plasmids and bacterial strains used in this study are listed in Table 1

Liquid chemically defined media for growth of S. agalactiae
The liquid chemically defined medium used to grow S. agalactiae (CDM) contains 8.

Measurement of bacterial growth
For measuring bacterial growth in chemically defined medium, S. agalactiae strains were first cultured in TH broth at pH 7.4 (without agitation) to the stationary phase of growth. These cultures were centrifuged and washed in non-buffered CDM at pH 7.4. They were then suspended to an OD 600 nm of 0.005 in the same medium and grown overnight at 37 °C, without agitation. These last cultures were finally diluted to an OD 600 nm of 0.05 in CDM adjusted to the pH and polyamine concentration of interest. These last cultures were incubated at 37 °C for 16 h in microtitre plates (Greiner Bio-One; Cellstar) (300 µl culture volume per well) in an Eon thermoregulated spectrophotometer plate reader (BioTek Instruments). The OD 600 nm was measured every hour after double orbital shaking of the plate for 5 s. The reported OD 600 nm is the average OD of three wells inoculated with the same culture. Three independent experiments were realized for all strains and for all tested conditions.
Prediction of transmembrane helices in PotB and PotC was performed by the TMHMM Server of the Center for Biological Sequence Analysis at the Technical University of Denmark (http://www. cbs. dtu. dk/ services/ TMHMM/).
The presence of a signal peptide and the location of its cleavage sites in PotD was predicted with the SignalP 5.0 server (http:// www. cbs. dtu. dk/ services/ SignalP/).

Survival of S. agalactiae to peroxidase-induced oxidative and acidic stress
To measure the ability of S. agalactiae strains to survive in CDM at pH 4.0, the bacteria were cultured at 37 °C (without agitation) in non-buffered TH broth at pH 7.4 to the beginning of the stationary phase of growth. This culture was then centrifuged, washed with non-buffered CDM at pH 7.4, and suspended to an OD 600nm of 0.005 in 40 ml of the same medium. After an overnight incubation at 37 °C without agitation, 10 ml aliquots of this culture were transferred to Falcon tubes, which were centrifuged for 5 min at 5000 g. Bacterial pellets were suspended in either 1 ml CDM buffered at pH 4.0 with a 100 mM mix of Na citrate and citric acid without the presence of polyamines or in 1 ml of the same medium containing 1 mM spermidine, spermine or putrescine. These suspensions were then incubated at 37 °C (without agitation) for 6 h. Viable cell counts of the bacteria were performed immediately after suspension of the pellets (t 0 ) and at suitable time intervals thereafter. To this end, serial dilutions were performed in TH broth at pH 7.4. Then 100 μl of each of these dilutions was immediately spread three times onto TH agar plates, which were incubated at 37 °C for 24 h. All survival experiments were performed at least three times. Results are expressed as the percentage of survivors [(number of viable bacteria at the tested condition divided by the number of viable bacteria at t 0 )×100].
To compare the sensitivity of the potABCD mutant and the wild-type strains to peroxidase-induced oxidative stress, S. agalactiae strains were grown to an OD 600 nm of 0.6 in TH broth and then exposed to different concentrations of H 2 O 2 (1, 5 or 20 mM).
To test if polyamines are involved in the survival of S. agalactiae strains submitted to peroxidase-induced oxidative stress, S. agalactiae cultures were grown in TH broth to the beginning of the stationary phase. Each culture was then centrifuged, washed with non-buffered CDM at pH 7.4, and suspended to an OD 600 nm of 0.005 in 10 ml of the same medium and grown overnight at 37 °C without agitation. These last cultures were finally diluted to an OD 600 nm of 0.05 in CDM at pH 7.4, and grown until an OD 600 nm of 0.6 (exponential phase). Bacterial cultures (10 ml samples) were then harvested and exposed to peroxidase-induced oxidative stress after suspension in the same volume of CDM containing either 5

Expression of potABCD during acidic and peroxidase-induced oxidative stress
To quantify the expression of the potABCD operon during acidic stress, strain A909 was grown in Falcon tubes containing 10 ml TH broth to the beginning of the stationary phase of growth. Each culture was then centrifuged, washed with non-buffered CDM at pH 7.4, suspended to an OD 600 nm of 0.005 in 10 ml of the same medium, and grown overnight at 37 °C without agitation. These last cultures were finally diluted to an OD 600 nm of 0.05 in CDM at pH 7.4, and grown to an OD 600 nm of 0.6 (exponential phase). Bacterial cells (10 ml samples) were then harvested, centrifuged, and suspended either in 10 ml CDM buffered at pH 5.5 with 100 mM MES or in CDM buffered at pH 4.0 with a mix of Na citrate and citric acid. These media were either supplemented or not with 1 mM spermidine, spermine or putrescine. The bacteria were exposed to the acidic stress for 30 min. A control culture grown at pH 7.4 in CDM without acidic stress was treated similarly. All cultures were then centrifuged at 5000 g. Bacterial pellets were collected and then stored at −80 °C until RNA extraction. This experiment was repeated three times from three independent cultures.
To determine the expression of potA during peroxidaseinduced oxidative stress by quantitative reverse transcriptase (qRT-PCR), the same treatment was performed as described for the peroxidase-induced oxidative stress survival assay. Thus, the wild-type cultures were collected after either 20 or 60 min of exposure to 5 mM H 2 O 2 , in the absence and in the presence of each type of polyamine. A control culture grown at pH 7.4 in CDM without any stress was always collected at the same time. Cultures were then centrifuged at 5000 g. Bacterial pellets were collected and then stored at −80 °C until RNA extraction. This experiment was repeated three times from three independent cultures.

Nucleic acid manipulations
Standard nucleic acid manipulation techniques were carried out as described previously [45]. S. agalactiae genomic DNA and RNA purifications were performed as previously described [40].  [46,47].

Amplification of nucleic sequences by PCR, by RT-PCR and by qRT-PCR
PCR was carried out with the Applied Biosystem 2720 Thermal cycler using Q5 High-Fidelity DNA polymerase (New England Biolab) for cloning or sequencing or OneTaq polymerase (New England Biolab) for analytical PCR. For cloning or sequencing, the resulting PCR fragments were further purified with a NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel) or with a NucleoSEQ kit (Macherey-Nagel), according to the manufacturer's instructions. The oligonucleotides (Sigma-Aldrich) used in this study are listed in Table 2.
For RT-PCR and qRT-PCR, the RNAs were reverse transcribed as previously described [40]. For RT-PCR, cDNAs were amplified by PCR with appropriate primers (Fig. 2a,  Table 2). The fold change in the transcript level was calculated using the following equations: ΔCt=Ct (target gene) -Ct (recA gene); ΔΔCt=ΔCt (reference condition) -ΔCt (test condition); relative quantification (RQ)=2 −ΔΔCt . Each assay was performed in triplicate and repeated with at least three independent RNA samples.

DNA sequencing
PCR products were sequenced on both strands using the Big Dye Terminator v3.1 cycle sequencing kit from Applied Biosystems and the ABI Prism 310 Genetic Analyzer.

Construction of potABCD deletion mutant
S. agalactiae A909ΔpotABCD is a non-polar mutant of strain A909 deleted by allelic exchange of a DNA region beginning five nucleotides after the stop of murB and ending 338 nucleotides after the start of potD. Upstream and downstream regions of the deleted region were amplified by PCR with primers OAH295 fw /OAH305 rv and OAH306 fw / OAH307 rv , respectively. These amplified fragments were cut by BsaI, and a recombination cassette, consisting of a fusion between these two regions, was obtained by splicing-by-overlap-extension PCR with primers OAH295 fw and OAH307 rv ( Table 2). To carry out chromosomal gene inactivation, the overlap-extension fragment was hydrolysed by BamHI and KpnI, and cloned into the BamHI/KpnI sites of the thermosensitive shuttle plasmid pG +host1 [48]. The recombinant plasmid was electroporated in E. coli for amplification, purified and finally electroporated in strain A909. Allelic exchange was performed as described by Biswas [48]. Deletion of the potABCD region of S. agalactiae A909 was confirmed by sequencing with primers SK3 fw , SK5 rv , SK6 fw and SK8 fw ( Table 2).

Determination of the intracellular polyamine content
Intracellular polyamine content was determined as described in the literature [49,50]. In brief, aliquots from bacterial cultures were pelleted. Then, 200 mg (wet weight) of bacteria was washed four times with PBS and suspended in 1 ml of 0.2 M perchloric acid. They were subsequently disrupted by sonication and centrifuged for 10 min at 12 000 g (4 °C

Statistical analyses
Data are presented as the mean±sd for three independent experiments. An unpaired Student's t-test was used to determine the significance of the differences between means [51].

RESULTS AND DISCUSSION
The putrescine, spermidine and spermine biosynthesis pathways are not present in S. agalactiae We searched, by blastP analysis, if S. agalactiae possesses homologues of enzymes involved in the known putrescine, spermidine and spermine biosynthesis pathways (Fig. 1).
Only three enzymes (aspartate kinase, aspartate β semialdehyde dehydrogenase and l-methionine adenosyl transferase) involved in one or the other first steps of spermidine or spermine biosynthesis were encoded by all strains of S. agalactiae. These three enzymes are also involved in the biosynthesis of several amino acids and are essential for S. agalactiae (e.g. SAK_0414, SAK_0954 and SAK_1141 in https://www. genome. jp/ kegg-bin/ show_ organism? org= sak). One (DK-PW-092) of the 1018 analysed strains also encodes an agmatine deiminase (gene WA34_16675) and an N-carbamoylputrescine amidohydrolase (gene WA34_ 16685). In the genus Streptococcus, the presence of polyamine biosynthesis pathways was searched by others in three other species. All the genomes of S. pneumoniae and the two completely sequenced genomes of Streptococcus oralis encode the enzymes necessary for the polyamine biosynthesis pathways IIp, Is and IIs (Fig. 1). However, these three pathways were found in only two of all the 30 completely sequenced genomes of S. suis and in two of the three completely sequenced genomes of S. mitis [11,19]. It is not known if the need of certain streptococcal species for a polyamine biosynthesis pathway reflects their lifestyle and their ability to survive at certain stages in polyamine-free environments.
As homologues of most of the enzymes involved in the known biosynthesis pathways of putrescine, spermidine and spermine are absent in S. agalactiae, this bacterium should not be able to synthesize these polyamines, and presumably acquired them from the environment. We verified this hypothesis by TLC analyses performed on the intracellular polyamine content of bacteria grown in the absence or in the presence of polyamines. Fig. 3(a) shows that S. agalactiae is unable to biosynthesize spermine, spermidine or putrescine (lane 1a), whereas it is able to strongly transport spermidine and spermine (lanes 2a and 3a) and slightly transport putrescine (lane 4a, white arrow) at pH 7.4.

Prevalence of the potABCD genes of S. agalactiae and characteristics of the transporter
As almost all bacteria can import extracellular polyamines by the ABC transporter potABCD, we searched the prevalence of the potABCD genes in S. agalactiae strains. To that end, blastN analyses with the nucleotide sequence of potABCD of strain A909 were performed in the non-redundant nucleotide collection of S. agalactiae sequences, available at the NCBI database. This operon is conserved in all the completely sequenced genomes of S. agalactiae (130 strains at the time of our analysis). We also screened this databank by tblastN with the PotA, PotB, PotC and PotD protein sequences of strain A909. In 90.8, 93.1, 97.0 and 99.2% of these strains, the PotA, PotB, PotC or PotD sequences are identical to those of strain A909, respectively. The few non-identical PotA, PotB or PotC proteins were translated from nucleotide sequences of pseudogenes. Non-identical PotA proteins contain only one mismatch with respect to the sequence of strain A909. Seventeen per cent of these mismatches concern similar amino acids.
The amino acid sequence of PotA has the characteristic motifs of the nucleotide binding proteins [52].  [52]. However, no lipid attachment site could be identified in PotD. We also searched for the presence of other domains involved in the anchoring of proteins to the surface of Gram-positive bacteria: LPXTG sequences for binding to the peptidoglycan by a sortase or choline-binding domains for the attachment to choline residues of lipoteichoic acids. None of them could be identified in PotD. This protein may thus be anchored to the cytoplasmic membrane by an as yet unidentified mechanism. A similar situation was described for PotD of S. pneumoniae [53]. PotD of S. agalactiae possesses the bacterial spermidine/putrescine binding motif described by Shah and collaborators [54]. This binding fold is composed of two globular subdomains connected by a flexible hinge and bind their ligand in the cleft between these domains in a manner resembling a Venus flytrap [55].

Transcriptional analysis of the potABCD region of S. agalactiae A909
The potABCD genes of S. agalactiae are transcribed in the same direction as the fol genes, murB and clc, suggesting that they are co-transcribed (Fig. 2a). Most of these genes are separated  Table 2, Fig. 2a), and a forward primer annealing in murB (SK6 fw , Table 2, Fig. 2a), we here showed that murB is also co-transcribed with potABCD (Fig. 2, lanes 2b and  5b, respectively). Similarly, by using the SK9 reverse primer annealing in clc and the SK8 forward primer annealing in potD ( Table 2, Fig. 2a), we showed that potD is co-transcribed with clc (Fig. 2, lane 8b). We previously showed that folK is co-transcribed with murB in strain SA87 [40]. By using the SK17 reverse primer annealing in murB and the SK16 forward primer annealing in folK ( Table 2, Fig. 2a), we confirm this fact in strain A909 (Fig. 2, lane 10b). As co-transcription of folK and murB and of potD and clc were demonstrated, the role of the two hairpin structures as transcriptional terminators is questionable; these structures could perhaps be attenuators or binding sites for a regulator protein. It is of note that an attenuator structure was identified in the leader sequence of the potABCD operon of Haemophilus somnus  2a, 1b, 1c and 1d), 1 mM spermine (lanes 3a, 2b, 2c and 2D) or 1 mM putrescine (lanes 4a, 3b, 3c and 3d). In (c) and (d), bacteria were then exposed to 5 mM H 2 O 2 for 60 min Bacterial extracts were dansylated, separated by TLC and photographed under Wood light. Dansylated standards of spermidine (0.2 µg; lanes 5a, 4b, 4c and 4d), spermine (0.2 µg; lanes 6a, 5b, 5c and 5d) and putrescine (0.1 µg; lanes 7a, 6b, 6c and 6d) were deposited in each TLC plate. The white arrows indicate the intracellular presence of a small quantity of putrescine, spermine or spermidine.
and of Pasteurella multocida [56]. We have thus compared the level of folK and of potD transcripts with those of folK-murB and potD-clc co-transcripts. To that end, RT-PCR was performed with reverse and forward primers annealing in folK (SK33 rv and SK32 fw ) and in potD (SK30 rv and SK29 fw ) ( Table 2, Fig. 2a). Although this experiment is rather qualitative, amplification of the folK-murB (Fig. 2, lane 10b) and potD-clc (Fig. 2, lane 8b) co-transcripts is lower than that of folK (Fig. 2, lane 2c) or potD (Fig. 2, lane 2d), suggesting that the two hairpin structures arrest some of the upstream transcripts.
In conclusion, although the murB-potABCD and the clc genes are also likely transcribed from their own P murB and P clc promoters, the above results suggest that strain A909 synchronizes the biosynthesis of folate and peptidoglycan with the transport of a polyamine by PotABCD and with the resistance to acidic stress. An RNA-sequencing experiment conducted on strain NEM316 of S. agalactiae indicated that the P murB and the P clc promoters are functional and that the murB-potABCD and clc transcripts are the major transcripts of the folK-clc region [57]. As the murB and potABCD genes are also co-transcribed in S. pneumoniae and S. suis, it appears that it is particularly important for streptococcal species to coordinate these two processes involved in cell wall synthesis [19,41]. It is nevertheless of note that although the synteny of the murB and potABCD genes is conserved in the entire genus Streptococcus, this is not the case for the complete folK-murB-potABCD-clc region. We compared the synteny of this region in the genomes of 676 streptococal strains representative of the genus Streptococcus and found that the entire folK-clc region is only conserved in species belonging to the pyogenic group (S.  . These cultures were incubated for 16 h at 37 °C without agitation in microtitre plates (300 µl culture volume per well) in an Eon thermoregulated spectrophotometer plate reader. The OD 600 nm was measured every hour after double orbital shaking of the plate for 5 s. The reported OD 600 nm is the average OD of three wells inoculated with the same culture. Three independent experiments were realized for all tested conditions. Standard deviations were always less than 10 %.

S. pasteurianus).
In the non-pyogenic species S. suis, the synteny is only conserved from potA to clc. However, Liu and collaborators were not able to show a co-transcription between potABCD and clc in this species [19] .
No requirement of polyamines and of the PotABCD transporter for the growth of S. agalactiae at pH 7.4 Since S. agalactiae seems to be obligated to acquire polyamines from the environment, we tested if the PotABCD transporter and exogenous polyamines are required to sustain growth of this bacterium. We first grew S. agalactiae strain A909 in a chemically defined medium containing various concentrations of spermine, spermidine or putrescine. As polyamines are very basic components, this medium was buffered at pH 7.4 with 100 mM HEPES. Concentrations of spermidine or spermine of 1 mM or below have only a very slight positive impact on the growth of S. agalactiae, but higher concentrations have an inhibitory impact (Fig. 4a,  b). Putrescine has no effect on the growth of S. agalactiae until a concentration of 5 mM, when it becomes slightly inhibitory (Fig. 4c). We then constructed a deletion mutant of potABCD to ensure that traces of polyamines eventually contaminating our minimal medium are not transported by the PotABCD transporter, thus allowing an identical growth of the wild-type strain in the presence or not of each of the polyamines tested. This assumption is, however, invalid as strains A909 and A909∆potABCD grow identically either in the rich TH medium or in the chemically defined medium in the absence of added polyamines (Fig. S1a, b, available in the online version of this article). No difference between the growth of the wild-type strain and of the potABCD deletion mutant in a chemically defined medium containing 1 mM spermidine, 1 mM spermine or 1 mM putrescine were also noted ( Fig. S1c-e, respectively). Furthermore, TLC analyses indicate that both the wild-type strain and the ΔpotABCD mutant efficiently import spermidine and spermine and putrescine faintly (Fig. 3a, b, respectively). Therefore, the potABCD transporter is not the main and only importer of polyamine in S. agalactiae.
In conclusion, S. agalactiae does not have an absolute need of exogenous polyamines in its environment, does not encode any specific enzymes involved in the synthesis of polyamines, has an undetectable content of intracellular polyamines when grown in the absence of polyamines, and does not need the PotABCD transporter for efficient in vitro growth in physiological conditions at pH 7.4. Bacterial species thus have different comportments with respect to polyamine requirement because, for optimal growth, S. pneumoniae, in which the polyamine biosynthesis pathways were inhibited, or strains of S. suis devoid of the polyamine biosynthesis pathways IIp, Is and IIs, show delayed growth after the deletion of genes encoding the PotABCD transporter [19,58]. Supplementation of growth media with polyamines was also shown to enhance the growth of certain bacteria such as E. coli mutants, which cannot synthesize polyamines, or Legionella pneumophila, which does not possess the enzymes required for polyamine biosynthesis [59,60].
Exogenous polyamine supplementation greater than 1 mM inhibited S. agalactiae in a dose-dependent manner ( Fig. 4 and data not shown). The bactericidal effect of certain concentrations of polyamines was reported for several bacterial species, and particularly in Staphylococcus aureus, which is hypersensitive to these compounds, even at a physiological concentration [19,61]. The reason for this inhibitory effect is not completely understood. Several reports have described a relationship between the toxicity of polyamines towards Staphylococcus aureus and an increase of pH of the medium. It was suggested that polyamine toxicity is inversely proportional to the net cationic charge of polyamines since they become sequentially deprotonated at elevated pH [61,62]. It was also found that Staphylococcus aureus polyamine sensitivity is mediated by menaquinone but is independent of respiration [61]. In E. coli, to avoid spermidine toxicity, high concentration of spermidine inhibit the polyamine ABC transporter, through the interaction of spermidine with PotA [28]. In addition, PotD is a retroactive regulator of the transcription of potABCD [63]. It is not known if these mechanisms exist and are efficient in S. agalactiae.

Spermine and spermidine induce the transcription of potABCD
We tried to correlate the transport of polyamines with the level of transcription of the potABCD operon. To that end, we quantified potA mRNA by qRT-PCR during growth of the bacteria in a chemically defined medium containing or lacking 1 mM spermine, spermidine or putrescine. As shown in Fig. 5, spermidine and spermine induce the expression of potABCD, during both the exponential (induction factor of 2.80 and 2.96, respectively) and the stationary phase (induction factor of 3.55 and 3.64, respectively) of growth. In contrast, putrescine does not have this effect. These data are in agreement with the intracellular polyamine content of cells grown in media with spermine, spermidine or putrescine (Fig. 3a).
Similarly, in S. suis, spermine and spermidine were found to induce the expression of the potABCD operon, but related experiments in S. pneumoniae gave different results as the expression of potD was found to be down-regulated in the presence of spermidine but up-regulated in the presence of putrescine in a low choline medium [4,19,41].

Influence of polyamines and PotABCD on the resistance of S. agalactiae to acidic pH
The co-transcription of potABCD with clc and the role of polyamines as key mediators in the resistance to acidic stress of some bacteria could indicate a role of polyamines and of potABCD in the acid resistance of S. agalactiae. We thus compared the growth of the wild-type strain S. agalactiae A909 and the mutant A909∆potABCD at the pH of two of its niches (pH 5.5, pH of the intestine; and pH 4.0, pH of the vagina). To this end, these strains were grown in a chemically defined medium supplemented or not with 1 mM spermidine, spermine or putrescine. As polyamines are very basic molecules, these media were buffered at pH 5.5 with 100 mM MES or at pH 4.0 with a 100 mM mix of Na citrate and citric acid to avoid modifications of their pH after the addition of polyamines. Our results revealed no significant difference between the wild-type cells and the mutant at all the tested conditions at pH 5.5 (Fig. S2). As no growth of S. agalactiae strains could be obtained at pH 4.0, either in the absence or in the presence of polyamines (results not shown), we compared the ability of the two strains to survive at pH 4.0. To this end, strains A909 and A909∆potABCD were incubated in the above cited media and the proportion of surviving bacteria was monitored over time. No significant difference in the survival capacity of the mutant A909 ΔpotABCD in comparison to the wild-type strain was visible, either in the absence or in the presence of polyamines (Fig. S3). We then compared the expression of the potABCD operon at pH 7.4, 5.5 and 4.0 by qRT-PCR. Our results show that acidic stress at pH 5.5 or at pH 4.0 have no effect on the expression of potA, either in the absence or in the presence of polyamines (Fig. 6a, b). These expression data correlate with phenotypic observations, since deletion of the PotABCD transporter had no effect on the growth or the survival of S. agalactiae under acidic conditions (Figs S2 and S3). However, TLC analyses indicate that both the wild-type strain and the ΔpotABCD mutant import spermidine and spermine efficiently and putrescine very faintly at pH 5.5 (Fig. S4). Hence, the PotABCD transporter is not involved in the resistance of strain A909 of S. agalactiae to acidic stress. However, this property is probably strain-dependent as a microarray analysis of strain 2603 V/R showed that transcription of the murB-potABCD operon (but not of the clc gene) is increased at pH 5.5 relative to that at pH 7.0 [63]. This induction was found to be dependent of the CsrRS two-component system (CovRS system), which is the major acid response regulator in that organism [64,65].

Influence of polyamines and PotABCD on the resistance of S. agalactiae to peroxide-induced oxidative stress
In several bacterial species, polyamines, by their function as radical scavengers, were implied in the protection from the toxic effects of reactive oxygen, so we tested if PotABCD has a role in this mechanism in S. agalactiae [2-4, 66, 67]. To this end, exponentially growing potABCD deletion mutant and wild-type strains were exposed to different concentrations of exogenous H 2 O 2 (1, 5 and 20 mM). The proportion of surviving bacteria was monitored over time, by plate counts. No significant differences in survival rate were obtained between strain A909 and A909ΔpotABCD (Fig. S5). We next tested if polyamines are involved in the survival of the same strains submitted to an oxidative stress of 5 mM H 2 O 2 . Again, the wild-type and the mutant strains died at the same rate, whether the growth medium was supplemented or not with spermidine, spermine or putrescine (Fig. S6). We then compared the expression of the potABCD operon by qRT-PCR in the absence or in the presence of H 2 O 2 . Hydrogen peroxyde significantly induced the expression of the potABCD operon by 2-fold after 20 min of incubation and by 3.4-fold after 60 min of incubation (Fig. 6c). The presence of polyamines in the medium during a peroxidase-induced oxidative stress of 60 min again enhanced the expression of this operon by 5.2-, 6.0-or 6.4-fold in the presence of spermidine, spermine or putrescine, respectively (Fig. 6d).
The above expression data suggest an involvement of PotABCD and polyamines in the resistance of S. agalactiae to peroxidase-induced oxidative stress. These data were confirmed by TLC analyses of the intracellular content of cells grown in the presence of polyamines during oxidative stress. In these conditions, the wild-type strain imports spermidine and spermine but also, now, a noticeable amount of putrescine (Fig. 3c). By contrast, only a small quantity of spermine and spermidine are transported by the ΔpotABCD mutant (Fig. 3d, white arrows). The absence of visible phenotypic effects after deletion of the potABCD operon should be explained by this slight transport of polyamines by as yet unidentified polyamine transporter(s). In S. pneumoniae, it was also shown that the potABCD operon is induced during a peroxidase-induced oxidative stress. The S. pneumoniae potABCD deletion mutant had nevertheless a comparable survival rate to the wild-type strain under exposure to the oxidizing stress-inducing agent paraquat [4,54]. However, different situations exist in the bacterial world as polyamine-deficient mutants of E. coli are killed in the presence of concentrations of oxygen that are non-toxic to wild-type cells [3].

Conclusion
In several bacterial species, PotABCD is able to transport different types of polyamines with different affinities. In a pioneering work on E. coli, Kashiwagi concluded that the order of preference of PotABCD is first putrescine, then spermidine and finally spermine [68]. However, this order of preference seems to vary between species [19,41,69]. In S. agalactiae, depending on the environment of the bacteria, the expression of potABCD is induced by different types of polyamines. The import of each of these polyamines thus appears to depend on their affinity towards PotABCD but also on the availability of this transporter, both being governed by the living environment of the bacteria. The suspected redundancy of some polyamine transporters of Streptococcus species was suggested to mask some of their phenotypic traits [4,54]. A similar situation exists also in S. agalactiae, making the analysis of these important transporters yet more complex.